Stem cells develop into several different cell types. Primarily, stem cells fall under two categories: embryonic and adult. In a developing embryo, stem cells differentiate into specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing specialized cells, but also maintain the normal turnover of regenerative organs, such as blood, skin, or intestinal tissues. Adult stem cells can differentiate into multiple pathways. Mesenchymal stem cells are adult stem cells which give rise to a variety of cell types including, bone cells (osteocytes), cartilage cells (chondrocytes), fat cells (adipocytes), and connective tissue (i.e., tendons). Neural stem cells are adult stem cells in the brain which give rise to its three major cell types: neurons; astrocytes and oligodendrocytes. Epithelial stem cells are adult stem cells in the lining of the digestive tract occur in deep crypts. Skin stem cells are adult stem cells which occur in the basal layer of the epidermis and at the base of hair follicles.
Hematopoietic stem cells (HSCs) are pluripotent, asymetrically self-renewing cells that give rise to all mature blood cells through successive rounds of differentiation. Specifically, HSCs have been identified in fetal bone marrow, fetal liver, umbilical cord blood (UCB), adult bone marrow, and peripheral blood following treatment with recombinant human granulocyte colony-stimulating factor (G-CSF), which are capable of differentiating into three cell lineages including myeloerythroid (i.e., red blood cells, granulocytes, monocytes), megakaryocyte (i.e., platelets) and lymphoid (i.e., T-cells, B-cells, and natural killer) cells. These HSCs are used in clinical transplantation protocols to treat a variety of diseases including malignant and non-malignant disorders. Currently, HSC transplantation is used to treat hematological diseases, such as leukemia and bone marrow failure. Expansion of HSCs would improve transplantation outcomes and help meet the demand for stem cell transplants by permitting the use of samples of limited quantity (e.g., cord blood) or with low total numbers of HSCs (e.g., poor HSC mobilizers). However, the relative inability to expand HSCs imposes a major limitation on the current use of HSC transplantation, and thus there is a shortage of HSCs for patient treatments.
The signals that govern the self-renewal process have been intensively pursued in order to facilitate HSC expansion by transiently enforcing proliferation pathways or blocking differentiation cues, which would be highly desirable as a means to augment the number of HSCs for transplantation. Human studies reveal that adult HSCs can undergo repeated rounds of asymmetric self-renewal with maintenance of the stem cell pool but with little or no expansion. In contrast, fetal and neonatal stem cells can be maintained in culture for 2-3 months with absolute increases in the number of HSCs. See Ando, K. et al., Blood. 2006; 107(8):3371-3377; and Dahlberg, A. et al., Blood. 2011; 117(23):6083-6090.
Homeobox (HOX) genes encode transcription factors that regulate patterning during embryonic development, and hematopoiesis both pre- and post-natally. In early development, HOX gene expression is both temporally and spatially regulated, as reflected by the sequential order of transcription with respect to their 3′ to 5′ chromosomal position. However, the spatio-temporal regulation of HOX gene expression is not observed in hematopoiesis, but instead assumes a complex, overlapping expression pattern that is not lineage-specific. Certain HOX genes, are highly expressed in HSCs and progenitor cells, and are generally downregulated as cells terminally differentiate into mature, lineage-specific blood cells. See Sauvageau, G et al., Proc Natl Acad Sci USA. 1994; 91(25):12223-12227. However, to date, the mechanisms underlying transcriptional regulation of HOX genes during hematopoiesis remain largely unknown.
The identification of HOX genes that are highly expressed in CD34+ HSCs and early progenitors led to the discovery that HOXB4 expression by retroviral transduction promoted the selective expansion of murine HSCs in cell culture and following bone marrow transplantation. See Sauvageau, G et al., Genes Dev. 1995; 9(14):1753-1765. Moreover, the enhanced proliferation of HOXB4-transduced HSCs did not alter HSC differentiation. See Thorsteinsdottir, U. et al., Blood. 1999; 94(8):2605-2612; and Antonchuk, J. et al., Cell. 2002; 109(1):39-45.
Conversely, when other HOX genes, such as HOXA9 were constitutively expressed in HSCs instances of myeloid leukemia developed. See Kroon, E. et al., Embo J. 1998; 17(13):3714-3725; and Care, A. et al., 1999; 18(11):1993-2001. Moreover, when the effects of HOX protein overexpression in different species were analyzed species-specific variations in the magnitude of HOX-induced HSC proliferation were observed. Specifically, modest effects observed in cells from humans and baboons when compared to those of mice and dogs. See Zhang, X B et al., J Clin Invest. 2008; 118(4):1502-1510. Additional studies revealed the development of myeloid leukemia in large mammals two years post-transplantation with HSCs expressing retrovirally-transduced HOXB4, highlighting the risks of such genetic manipulation. See Zhang, X B., J Clin Invest. 2008; 118(4):1502-1510. Taken together, current research indicates that ectopic HOX expression is not likely to produce an HSC expansion method capable of promoting HSC proliferation at a level necessary to create therapeutically significant amount of HSCs.
Therefore, a transient delivery method to augment HOX expression levels in HSCs is necessary for potential human therapeutic applications. To that end, direct protein transduction methods resulted in comparable levels of ex vivo murine HSC proliferation when compared with retroviral integration, but the short half-life of HOX protein necessitated frequent replenishing of the recombinant protein, which is impractical for large-scale ex vivo expansion of HSCs. See Krosl, J. et al., Nat Med. 2003; 9(11):1428-1432. Recently, studies have sought to stabilize the HOXB4 protein by modifying the N-terminal domain of the native peptide. See U.S. Pat. No. 8,039,463. However, while mutations in the N-terminal resulted in an increase in protein stability, such mutants were incapable of outcompeting wild-type HOXB4 protein, resulting in reduced long-term repopulation capabilities.